Method for analyzing activated polyethylene glycol compounds

ABSTRACT

A chemical analysis method for the determination of RO(CH 2 CH 2 O) n H, RO(C 2 H 4 O) n A, and AO(C 2 H 4 O) n A in a mixture thereof, wherein R is an alkyl group, A is a functional group for coupling with a surface or a biologically active material or another thing of use and n is an integer greater than 10. The method includes the derivatizing the A groups of the mixture with a derivatizing agent to form a derivatized mixture comprising RO(CH 2 CH 2 O) n H, RO(C 2 H 4 O) n AD, and DAO(C 2 H 4 O) n AD, wherein AD is the derivatized A group. The second step is chromatographing a sample of the derivatized mixture by liquid chromatography under critical conditions to determine the relative amounts of RO(CH 2 CH 2 O) n H, RO(C 2 H 4 O) n AD, and DAO(C 2 H 4 O) n AD in the derivatized mixture.

BACKGROUND OF THE INVENTION

The instant invention relates to chemical analysis methods for analyzingpolyethylene glycol compounds. More particularly, the instant inventionrelates to the analysis of polyethylene glycol compounds by liquidchromatography under “critical conditions”. The polyethylene glycolcompounds of the instant invention have been “activated” to facilitatechemical modification of physiologically active materials, whichmodified materials are applicable, for example, in drug deliverysystems.

Biologically active compounds conjugated with polyoxyalkylenes canprovide enhanced biocompatibility for the compound, See, for example,U.S. Pat. No. 5,366,735 and U.S. Pat. No. 6,280,745. A review of thissubject by Zalipsky, in Bioconjugate Chem., 1995, 6, p150-165,identified polyethylene glycol as one of the best biocompatible polymersto conjugate with a biologically active compound (such as a drug, aprotein, a peptide or an enzyme) to produce a conjugate having improvedproperties such as compatible solubility characteristics, reducedtoxicity, improved surface compatibility, increased circulation time andreduced immunogenicity.

Polyethylene glycol (PEG) is a linear polyoxyalkylene terminated at theends thereof with hydroxyl groups and generally represented by theformula: HO(CH₂CH₂O)_(n)H. Monomethoxy polyethylene glycol (mPEG) isgenerally represented by the formula: CH₃O(CH₂CH₂O)_(n)H. mPEG can be“activated” with a group “A” that will couple with a group of thebiologically active material. Activated mPEG is generally represented bythe formula: CH₃O(CH₂CH₂O)_(n)A. For example, trichloro-s-triazineactivated mPEG will couple to an amine group of a biologically activematerial, as discussed by Henmanson in Chapter 15 of BioconjugateTechniques (1996).

More recently, so called “second generation” PEGylation chemistry hasbeen developed to, for example, minimize problems of diol impuritycontamination of mPEG, to increase the molecular weight of the mPEG andto increase stability of the conjugate, see Roberts et al., AdvancedDrug Delivery Reviews 54 (2002) p459-4. U.S. Pat. No. 6,455,639described an increased molecular weight mPEG having narrow molecularweight distribution.

Liquid chromatography under critical conditions has become an importantmethod for polymer analysis, see, for example, Gorbunov et al., J. ChromA, 955 (2002) 9-17. Liquid chromatography under critical conditions hasbeen used to determine polyethylene glycol in mPEG (see, for example,Baran et al., J. Chrom. B, 753 (2001) 139-149; and Kazanskii et al.,Polymer Science, Series A, Vol 42, No. 6 (2000), p585-595. However, thedegree of resolution of the polyethylene glycol and mPEG peaks is poorwhen the molecular weight of the mPEG is 5,000 grams per mole or more(see FIG. 2 of the Kazanskii et al. reference). And, liquidchromatography under critical conditions has not been used to analyzeactivated mPEG.

SUMMARY OF THE INVENTION

The instant invention is the discovery that liquid chromatography undercritical conditions can be used to analyze activated mPEG for residualmPEG alcohol, activated mPEG and activated PEG diol even when themolecular weight of the mPEG is 5,000 grams per mole or more.Furthermore, derivatization of the activated mPEG and activated diolaccording to the instant invention can increase their chromatographicresolution. In addition, derivatization of the activated mPEG andactivated diol according to the instant invention can facilitatechromatographic detectability.

More specifically, the instant invention in one embodiment is a chemicalanalysis method for the determination of RO(CH₂CH₂O)_(n)H,RO(C₂H₄O)_(n)A, and AO(C₂H₄O)_(n)A in a mixture thereof, wherein R is analkyl group, A is a functional group for coupling with a surface or abiologically active material or an other thing of use and n is aninterger greater than 10, comprising the step of: chromatographing asample of the mixture by liquid chromatography under critical conditionsto determine the relative amounts of RO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)A,and AO(C₂H₄O)_(n)A in the mixture.

In another embodiment the instant invention is a chemical analysismethod for the determination of RO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)A, andAO(C₂H₄ 0)_(n)A in a mixture thereof, wherein R is an alkyl group, A isa functional group for coupling with a surface or a biologically activematerial or another thing of use and n is an interger greater than 10,comprising the steps of: (a) derivatizing the A groups of the mixturewith a derivatizing agent to form a derivatized mixture comprisingRO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)AD, and DAO(C₂H₄O)_(n)AD, wherein AD isthe derivatized A group; and (b) chromatographing a sample of thederivatized mixture by liquid chromatography under critical conditionsto determine the relative amounts of RO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)AD,and DAO(C₂H₄O)_(n)AD in the derivatized mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reproduction of a chromatogram showing the separation ofmPEG, propionaldehyde diacetal activated mPEG and propionaldehydediacetal activated diol;

FIG. 2 is a reproduction of a chromatogram showing the separation ofmPEG alcohol, mesylate activated mPEG and mesylate activated diol; and

FIG. 3 is a reproduction of a chromatogram showing the separation ofmPEG alcohol, para-nitrophenyl carbonate activated mPEG andpara-nitrophenyl carbonate activated diol.

DETAILED DESCRIPTION

The compounds to be analyzed by one method of the instant invention arerepresented by the formulas I, II and III:

(I) RO(C₂H₄O)_(n)A;

(II) AO(C₂H₄O)_(n)A; and

(III) RO(C₂H₄O)_(n)H

wherein R represents a C₁₋₇ hydrocarbon group (usually a methyl group),n represents the average number of moles of C₂H₄O groups, e.g., from 10to 2000 and A is the “activating” group. In many applications, thecompound of formula I is the desired material. The compound of formulaIII is non-activated PEG which is unreactive. The compound of formula IIis diactivated PEG which is produced from PEG diol impurity. Thus, thesample to be analyzed usually consists primarily of the compound offormula I with relatively lower levels of the compounds of formulas IIand III in a mixture. The relative amounts of the compounds of formulaI, II and III are determined by chromatographing a sample of the mixtureby liquid chromatography under critical conditions to determine therelative amounts of RO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)A, and AO(C₂H₄O)_(n)Ain the mixture. It should be understood that the instant invention inits full scope includes PEG copolymers (for example, random or blockcopolymers comprising C₂H₄O groups) and any polymer topology such aslinear, branched, comb and star topologies.

It should be understood that a certain degree of experimentation isrequired to achieve liquid chromatography under critical conditions.However, reference to the literature will direct the person of ordinaryskill in the art of liquid chromatography to the necessary conditions,see, for example, Gorbunov et al., J. Chrim A, 955 (2002) 9-17. Criticalcondition LC tends to separate polymers based on the composition of theend groups of the polymer and is (in theory) independent of the polymersmolecular weight. For example, if a reverse phase column is operatedunder critical conditions, then polymers with hydrophobic end groups canbe separated from polymers with hydrophilic end groups.

EXAMPLE 1

0.1 gram of propionaldehyde diacetal activated 5,000 weight averagemolecular weight mPEG is mixed with 3 milliliters of water to produce asample for injection. 5 microliters of the sample for injection isinjected into a moblile phase of 52% A and 48% B (where A is 47%acetonitrile in water and B is 43% acetonitrile in water) at a mobilephase flow rate of 0.75 milliliters per minute and flowed through a 5micrometer packing diameter Supelco LC-18 reverse phase column at acolumn temperature of 30 degrees Celsius, the column having an internaldiameter of 4.6 millimeters and a length of 250 millimeters, followed byan evaporative light scattering detector to produce the chromatogramshown in FIG. 1. The chromatogram of FIG. 1 shows a peak at about 3.8minutes for mPEG, a peak at about 4.8 minutes for the activated mPEG anda peak at about 5.6 minutes for the activated diol. The mole percentconcentration of mPEG is determined by dividing the peak area of themPEG peak by the combined peak areas of the mPEG, the activated mPEG andthe activated diol peak areas and then multiplying by 100. The molepercent concentration of activated mPEG is determined by dividing thepeak area of the activated mPEG peak by the combined peak areas of themPEG, the activated mPEG and the activated diol peak areas and thenmultiplying by 100. The mole percent concentration of activated diol isdetermined by dividing the peak area of the activated diol peak by thecombined peak areas of the mPEG, the activated mPEG and the activateddiol peak areas and then multiplying by 100.

Evaporative light scattering detection is well-known in liquidchromatography, see, for example, Rissler, J. Chrom. A, 742 (1996) 45.

EXAMPLE 2

0.1 gram of mesylate activated 5,000 weight average molecular weightmPEG is mixed with 3 milliliters of water to produce a sample forinjection. 5 microliters of the sample for injection is injected into amoblile phase of 52% A and 48% B (where A is 47% acetonitrile in waterand B is 43% acetonitrile in water) at a mobile phase flow rate of 0.75milliliters per minute and flowed through a 5 micrometer packingdiameter Supelco LC-18 reverse phase column at a column temperature of30 degrees Celsius, the column having an internal diameter of 4.6millimeters and a length of 250 millimeters, followed by an evaporativelight scattering detector to produce the chromatogram shown in FIG. 2.The chromatogram of FIG. 2 shows a peak at about 3.8 minutes for mPEG, apeak at about 4.4 minutes for the activated diol and a peak at about 4.9minutes for the activated mPEG. The mole percent concentration of mPEGis determined by dividing the peak area of the mPEG peak by the combinedpeak areas of the mPEG, the activated mPEG and the activated diol peakareas and then multiplying by 100. The mole percent concentration ofactivated mPEG is determined by dividing the peak area of the activatedmPEG peak by the combined peak areas of the mPEG, the activated mPEG andthe activated diol peak areas and then multiplying by 100. The molepercent concentration of activated diol is determined by dividing thepeak area of the activated diol peak by the combined peak areas of themPEG, the activated mPEG and the activated diol peak areas and thenmultiplying by 100.

EXAMPLE 3

0.1 gram of para-nitro phenyl carbonate activated 20,000 weight averagemolecular weight mPEG is mixed with 3 milliliters of water to produce asample for injection. 5 microliters of the sample for injection isinjected into a moblile phase of 52% A and 48% B (where A is 47%acetonitrile in water and B is 43% acetonitrile in water) at a mobilephase flow rate of 0.75 milliliters per minute and flowed through a 5micrometer packing diameter Jupiter C-18 reverse phase column at acolumn temperature of 29 degrees Celsius, the column having an internaldiameter of 4.6 millimeters and a length of 150 millimeters, followed byan evaporative light scattering detector to produce the chromatogramshown in FIG. 3. The chromatogram of FIG. 3 shows a peak at about 3minutes for mPEG, a peak at about 4.8 minutes for the activated mPEG anda small peak at about 8.5 minutes for activated diol. The mole percentconcentration of mPEG is determined by dividing the peak area of themPEG peak by the combined peak areas of the mPEG, the activated mPEG andthe activated diol peak areas and then multiplying by 100. The molepercent concentration of activated mPEG is determined by dividing thepeak area of the activated mPEG peak by the combined peak areas of themPEG, the activated mPEG and the activated diol peak areas and thenmultiplying by 100. The mole percent concentration of activated diol isdetermined by dividing the peak area of the activated diol peak by thecombined peak areas of the mPEG, the activated mPEG and the activateddiol peak areas and then multiplying by 100.

Derivatized Activated mPEG

In another embodiment, the instant invention is a chemical analysismethod for the determination of the above discussed RO(CH₂CH₂O)_(n)H,RO(C₂H₄ 0)_(n)A, and AO(C₂H₄ 0)_(n)A in a mixture thereof, comprisingthe steps of: (a) derivatizing the A groups of the mixture with aderivatizing agent to form a derivatized mixture comprisingRO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)AD, and DAO(C₂H₄O)_(n)AD, wherein AD isthe derivatized A group; and (b) chromatographing a sample of thederivatized mixture by liquid chromatography under critical conditionsto determine the relative amounts of RO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)AD,and DAO(C₂H₄O)_(n)AD in the derivatized mixture. This embodiment of theinstant invention is especially applicable when the activating group ishydrophilic, such as a group comprising, without limitation thereto, analdehyde, a maleimide, an amine or a thiol. mPEGs activated with ahydrophilic group can be difficult to separate from mPEG alcohol,because the hydroxyl group of the mPEG alcohol is also hydrophilic.However, it has been discovered that if such activated mPEG isderivatized with a derivatizing agent which attaches a hydrophobic groupto the A group of the activated mPEG, then the derivatized activatedmPEG can be more readily separated from the non-activated PEG alcohol.Similarly, mPEGs activated with a hydrophilic group can be difficult toseparate from PEG diols activated with two hydrophilic groups, becausethe methyl group of the mPEG alcohol is not very hydrophobic. However,it has been discovered that if the A groups of the activated mPEG andthe di-activated PEG are derivatized with a derivatizing agent whichattaches a hydrophobic group to the A groups on both the activated mPEGand the di-activated PEG, then the derivatized activated mPEG can bemore readily separated from the derivatized di-activated PEG.

Examples of suitable derivatizing agents include aromatic/aliphaticaldehydes/ketones for reductive amination of mPEG/PEG activated withamine; aromatic/aliphatic disulfides to convert mPEG/PEG thiols to mixeddisulfides; and aromatic/aliphatic hydrazines such as1-(hydrazinocarbonylmethyl)pyridinium chloride or dinitro phenylhydrazine) to convert mPEG/PEG activated with carbonyl groups to thecorresponding hydrazone group.

The derivatizing agent most preferably imparts a detectablecharacteristic, e.g. ultra-violet (UV) chromaphore or fluorescent group,to the derivatized activated mPEG/PEG to allow the derivatized activatedmPEG/PEG to be detected when it is eluted from the critical LC system.And, it should be understood that even when the activating group(s) Ahas(have) sufficient hydrophobic character to permit sufficientresolution of activated mPEG from di-activated PEG in the critical LCchromatogram, it may never-the-less be desirable to derivatize theactivated mPEG and di-activated PEG using a derivatizing agent thatimparts sufficient detectable characteristic to be detected when it iseluted from the critical LC system.

CONCLUSION

In conclusion, it should be readily apparent that although the inventionhas been described above in relation with its preferred embodiments, itshould be understood that the instant invention is not limited therebybut is intended to cover all alternatives, modifications and equivalentsthat are included within the scope of the invention as defined by thefollowing claims.

1-13. (canceled)
 14. A chemical analysis method for the determination ofRO(CH₂CH₂O)_(n)H, RO(C₂H₄O)_(n)A, and AO(C₂H₄O)_(n)A in a mixturethereof, wherein R is an alkyl group, A is a functional group forcoupling with a surface or a biologically active material or other thingof use and n is an integer greater than 100, comprising the steps of:(a) derivatizing the A groups of the mixture with a derivatizing agentto form a derivatized mixture comprising RO(CH₂CH₂O)_(n)H,RO(C₂H₄O)_(n)AD, and DAO(C₂H₄O)_(n)AD, wherein AD is the derivatized Agroup; and (b) chromatographing a sample of the derivatized mixture byreverse phase liquid chromatography under critical conditions todetermine the relative amounts of RO(C₂H₄O)_(n)AD, and DAO(C₂H₄O)_(n)ADin the derivatized mixture.
 15. The method of claim 14, wherein Rconsists essentially of a methyl group.
 16. The method of claim 14,wherein A comprises a carbonyl group.
 17. The method of claim 16,wherein A comprises an aldehyde group.
 18. The method of claim 17,wherein the aldehyde group is propionaldehyde.
 19. The method of claim16, wherein the derivatizing agent is1-(hydrazinocarbonylmethyl)pyridinium chloride.
 20. The method of claim16, wherein the derivatizing agent is dinitro phenylhydrazine.
 21. Themethod of claim 14, wherein A is maleimide.
 22. The method of claim 21,wherein the derivatizing agent is thio-naphthalene.
 23. The method ofclaim 14, wherein A comprises an amine.
 24. The method of claim 23,wherein the derivatizing agent is an aromatic aldehyde.
 25. The methodof claim 14, wherein A comprises a thiol group.
 26. The method of claim25, wherein the derivatizing agent is ortho dipyridynyl disulfide. 27.The method of claim 14, wherein the weight average molecular weight ofthe RO(C₂H₄O)_(n)A is greater than 5,000 grams per mole.
 28. The methodof claim 27, wherein the weight average molecular weight of theRO(C₂H₄O)_(n)A is greater than 10,000 grams per mole.